犬隻黑色素瘤和c-kit致癌基因之臨床相關性研究

Abstract

黑色素瘤(Melanoma)為犬的惡性腫瘤，致病的原因不明，對化療或放療反應不佳，目前有許多新療法如基因治療、標靶治療等正在研發。KIT是一個細胞膜上的RTK(Receptor tyrosine kinase)，藉由與其配體SCF(Stem cell factor)的接合，可活化細胞內的訊息傳遞，進而促進特定的細胞功能；若KIT的功能失調(過度表現或出現突變)則會使細胞癌化，在活體上造成惡性腫瘤。KIT的過度表現甚至是突變已在人黑色素細胞瘤中發現，尤其是在黏膜或肢端指甲下的黑色素瘤，其c-kit基因型的表現與標靶治療的效果具有相關性。本實驗以免疫組織化學染色法，佐以PCR技術，來分析臺灣大學動物醫院組織病理資料庫中49個犬黑色素瘤病例的KIT表現及c-kit基因突變的情形，再併同臨床上如發生的位置及預後等相關資料，分析其相關性，以評估KIT作為治療犬黑色素瘤標的之可行性。本研究蒐集的病例平均年齡為12±2.9歲，雄性佔59%(24/49)，雌性佔41%(20/49)，在可以評估的40個免疫組織化學染色的切片中，KIT染色皆為陽性，其中40%(16/40)呈現弱陽性，60%(24/40)為強陽性；在統計學上，KIT蛋白表現的強弱與犬黑色素瘤的發生位置、復發率或患犬的存活時間都沒有顯著的關聯性，但與腫瘤的轉移則有顯著的關聯性(p<0.05)；在49個檢體中，c-kit外顯子11皆沒有發現任何型式的內生性連續性複製(Internal tandem duplications; ITDs)突變，但在其中PCR產物的定序結果發現，一個C1743T的靜默突變(Silence mutation)存在於5個檢體中，這5個檢體中另發現一個T1736C的錯義突變(Missense mutation)，造成一個L579P的胺基酸突變，這個突變的發現，將有機會使用標靶藥物來治療部分犬的惡性黑色素瘤。

Melanoma is a malignant tumor in dogs, and can be found associated with skin, nail bed or oral cavity. The etiology of melanoma in dogs is largely unknown. Systemic chemotherapy for malignant melanoma in the dog has not shown any significant benefit. Coarse fraction radiotherapy has only been used for local control. However, most dogs with malignant disease succumb to systemic spread. Several novel therapeutic modalities have been investigated. These include methods of gene therapy, target therapy, etc. KIT is a receptor tyrosine kinase, and SCF(stem cell factor) is the ligand for KIT. SCF is capable of binding to KIT resulting in activation of intracellular domain and initiation of multiple downstream signal transductions. Mutation or overexpression of KIT can transform indolent tumors to aggressive tumors in vivo. In human, KIT have long been hypothesized to play key roles in melanoma development, especially in mucosal and acral melanomas. Importantly, expression of mutation was then found to correlate with the sensitivity of target therapy. In this study, there were 49 cases of canine melanomas collected from National Taiwan University Animal Hospital. KIT protein expression was determined by immunohistochemistry, and KIT gene mutations were analysed by PCR amplification and electrophoresis. Then we discussed the clinical relevance of canine melanomas and KIT, such as the location, prognosis, etc., and evaluate the possibility of the target therapy in canine melanoma. The average age of these 49 cases was 12±2.9 years old. In 40 slides of immunohistiochemistry, all the samples presented positive, then 40%(16/40) for weak positive and 60%(24/40) for strong positive. There were no statistical significances between KIT expression and canine melanoma location, local recurrence rate or survival times. However, these was statistical significance between KIT expression and canine melanoma metastasis rate. The stronger KIT expression, the higher metastasis rate was noted. In the PCR and electrophoresis results, there was no ITD (internal tandem duplications) found in exon 11 of c-kit. Otherwise, a C1743T silence mutation of exon 11 of c-kit was revealed in five canine oral mucosal melanomas by sequencing, and one of the five sample existed a T1736C in-frame missense (L579P) mutation. There is the possibility of target therapy for canine melanoma in the future.